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Identification of the Receptor Site for Triazine Herbicides in Chloroplast Thylakoid Membranes 1

KATHERINE Ε. STEINBACK, KLAUS PFISTER and CHARLES J. ARNTZEN Michigan State University, MSU-DOE Plant Research Laboratory, East Lansing, MI 48824

A broad range of inhibitors of photosynthesis act by blocking the same step in electron transport in chloroplast membranes. Inhibition occurs at the level of a protein-bound plastoquinone (B) that functions as the second stable electron acceptor for photosystem II (PS II). Studies based on the use of the proteolytic enzyme, tryp sin, indicate that the receptor site for PS II inhibitors is a protein of the structural PS II complex. Using a photoaffinity C-labeled derivative of atrazine, we have identified the specific receptor polypeptide for this inhibitor of PS II function. Analysis of membrane polypep tides by polyacrylamide gel electrophoresis and fluorographic techniques have shown that the photoaffinity triazine covalently binds to a polypeptide of 32-34 kilodaltons. We have fur ther shown that this polypeptide is surface expos ed; trypsin treatment of thylakoid membranes re sults in the stepwise alteration of the peptide to a 16 kilodalton species. The site for covalent attachment of the photoaffinity probe is located on the intrinsic 16 kilodalton fragment. Conse quently, the binding site appears to be deter mined, in part, by the intrinsic hydrophobic do main of the 32-34 kilodalton polypeptide. The biogenesis of the 32-34 kilodalton polypeptide is discussed with relation to genetic mechanisms that may be responsible for triazine resistance at the level of the chloroplast membrane. 14

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Current address: University of Wurzburg, Botanical Institute, Wurzburg 87, West Ger

many. 0097-6156/82/0181-0037$05.00/0 © 1982 American Chemical Society Moreland et al.; Biochemical Responses Induced by Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Approximately h a l f of a l l commercial h e r b i c i d e s act by i n h i b i t i n g photosynthesis by i n t e r a c t i n g with s p e c i f i c s i t e s along the photosynthetic e l e c t r o n t r a n s p o r t c h a i n . A number of d i v e r s e chemicals i n c l u d i n g the ureas, amides, t r i a z i n e s , t r i a z i n o n e s , u r a c i l s , p y r i d a z i n o n e s , q u i n a z o l i n e s , t h i a d i a z o l e s , and c e r t a i n phenols are thought to act s p e c i f i c a l l y at a common i n h i b i t o r y s i t e at the reducing s i d e of photosystem II (PS II) (1, 2 ) . Several l i n e s of evidence i n d i c a t e that t h i s i n h i b i t i o n occurs at the l e v e l of a p r o t e i n bound plastoquinone c a l l e d "B" (3). This e l e c t r o n c a r r i e r acts as the second s t a b l e e l e c t r o n acceptor of PS II (4^ 5 ) . It has been proposed that the common mode of a c t i o n of these chemical c l a s s e s i s v i a h i g h - a f f i n i t y binding to the PS II complex {6). Herbicide binding induces a change in the redox p o t e n t i a l of the quinone c o f a c t o r of B, thereby making the t r a n s f e r of e l e c t r o n s from Q (the f i r s t s t a b l e e l e c t r o n acceptor o f PS II) thermodynamically unfavorable (3, 5 ) . Establishment of s t r u c t u r e - a c t i v i t y r e l a t i o n s ï ï i p s within h e r b i c i d e c l a s s e s has been extremely useful in e l u c i d a t i n g important s t r u c t u r a l aspects of the i n h i b i t o r molecules themselves (_7). U n t i l r e c e n t l y , however, much l e s s emphasis has been d i r e c ted at understanding the biochemical c h a r a c t e r i s t i c s of the h e r b i c i d e receptor s i t e within the c h i o r o p l a s t membrane. Progress in t h i s d i r e c t i o n was i n i t i a t e d when Strotmann and T i s c h e r ( β , 8) introduced techniques f o r monitoring s p e c i f i c binding of h e r b i c i d e s to i s o l a t e d c h i o r o p l a s t t h y l a k o i d membranes. These and other workers (_3, j>, 9) have u t i l i z e d a v a r i e t y of r a d i o l a beled i n h i b i t o r s of PS II f u n c t i o n f o r the c h a r a c t e r i z a t i o n of p r o p e r t i e s of the h e r b i c i d e binding s i t e ; the studies have r e s u l ted in the demonstration of a competition f o r a s i n g l e binding domain per photosynthetic e l e c t r o n transport c h a i n . An understanding of the biochemical c h a r a c t e r i s t i c s of the PS II l o c a l i z e d h e r b i c i d e receptor domain is p a r t i c u l a r l y r e l e vant because of the appearance of t r i a z i n e - r e s i s t a n t weed biotypes in the United S t a t e s , Canada, and Europe (3). I n i t i a l attempts at understanding the mechanism(s) of resisTance d i r e c t e d i n v e s t i gators to evaluate a l t e r a t i o n s in uptake, t r a n s l o c a t i o n , or meta bolism of t r i a z i n e s . Only small d i f f e r e n c e s between s u s c e p t i b l e and r e s i s t a n t biotypes were e s t a b l i s h e d , these being i n s u f f i c i e n t to e x p l a i n the mechanism of extreme h e r b i c i d e r e s i s t a n c e . As the primary mechanism of action of the s - t r i a z i n e s i n v o l ves i n h i b i t i o n of PS II e l e c t r o n t r a n s p o r t , a t t e n t i o n was also d i r e c t e d at a n a l y s i s of c h i o r o p l a s t r e a c t i o n s in r e s i s t a n t weed biotypes (10, 11, 12). These studies can be summarized as f o l lows: (a) m ΤΓ1 cases studied to date, there is a m o d i f i c a t i o n in the c h i o r o p l a s t membranes of r e s i s t a n t biotypes that changes the c h a r a c t e r i s t i c s of s - t r i a z i n e b i n d i n g ; (b) t h i s m o d i f i c a t i o n r e s u l t s in a l t e r e d binding c h a r a c t e r i s t i c s of other c l a s s e s of h e r b i c i d e s , ( i . e . , only s l i g h t r e s i s t a n c e to ureas, but increased s e n s i t i v i t y to phenols) (see L3 f o r review), and (c) the a l t e r a t i o n of the h e r b i c i d e receptor in r e s i s t a n t weeds is accompanied

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by a d e t e c t a b l e change in the k i n e t i c c h a r a c t e r i s t i c s of e l e c t r o n t r a n s f e r from Q" to Β in the native membranes even 1n the absence of a l l h e r b i c i d e s (3^ 14, Jj>), which i n d i c a t e s that the apoprotein o f Β may be a l t e r e d i n T h e r e s i s t a n t c h l o r o p l a s t s so that the bound quinone c o f a c t o r e x h i b i t s d i f f e r e n t redox p r o p e r t i e s . In t h i s paper, we have summarized our current understanding of the biochemical nature of the t r i a z i n e binding s i t e within the PS II complex. Studies using the p r o t e o l y t i c enzyme t r y p s i n as a s e l e c t i v e , s u r f a c e - s p e c i f i c m o d i f i e r of membrane polypep t i d e s and the use of a p h o t o a f f i n i t y t r i a z i n e have been u t i l i z e d s e p a r a t e l y to i d e n t i f y the t r i a z i n e receptor protein as a 32-34 k i l o d a l t o n (kDal) polypeptide of the PS II complex in peas (Pisum sativum L . ) . The nature of the covalent attachment of the p h o t o a f f i n i t y probe has also enabled us to i d e n t i f y the t r i a z i n e receptor p r o t e i n as a product of c h l o r o p l a s t - d i r e c t e d p r o t e i n s y n t h e s i s ; t h i s implies that the s t r u c t u r a l gene f o r the t r i a z i n e receptor polypeptide i s encoded on c h i o r o p l a s t DNA. This i s in agreement with r e p o r t s , based on c l a s s i c a l genetic a n a l y s i s , that t r i a z i n e r e s i s t a n c e in B r a s s i c a campestris L. (16) i s a maternally i n h e r i t e d t r a i t . Materials

and Methods

Chioroplast Isolation. C h l o r o p l a s t s of peas, spinach ( S p i n a c i a o l e r a c e a L . ) , and biotypes of Amaranthus hybridus L. suscept i b l e or r e s i s t a n t t o _ s - t r i a z i n e s were i s o l a t e d and stroma-free t h y l a k o i d s prepared as p r e v i o u s l y described (17). Intact c h l o r o p l a s t s were obtained from pea leaves f o l l o w i n g the method of B l a i r and E l l i s (18). Trypsin Treatment. Trypsin incubations were c a r r i e d out at room temperature as p r e v i o u s l y described (JL9). Trypsin concen t r a t i o n s used are s p e c i f i e d in the t e x t . Photoaffinity Labeling. The p h o t o a f f i n i t y l a b e l i n g of pea thyla1 DCPIP). 14

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Figure 2B. Comparison of data derived from studies of C-atrazine affinity for thylakoid membranes incubated with trypsin with that obtained from electron transport assays (H 0 -> DCPIP) in the presence of 0.25 μΜ atrazine. 14

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p r o t e o l y t i c enzyme). This was followed by a gradual change in a f f i n i t y over the next 15 min of treatment. This i s in c o n t r a s t to the gradual time-dependent decrease in binding s i t e s , implying at l e a s t a two-step a l t e r a t i o n in the p r o t e i n (or p r o t e i n s ) of the PS II complex that c o n s t i t u t e the a t r a z i n e binding s i t e . Mild t r y p s i n treatment has been shown also to a l t e r the a f f i n i t y f o r a number of other chemical f a m i l i e s of PS II d i r e c ted h e r b i c i d e s in a manner s i m i l a r to that of the s - t r i a z i n e s . Trypsin-mediated decreases in i n h i b i t o r y a c t i v i t y are found for u r a c i l (19), urea, pyridazinone ( 1 £ , 28) and t r i a z i n o n e (28) herbicides. In c o n t r a s t , p h e n o l - t y p e T e r b i c i d e s increasecTin i n h i b i t o r y a c t i v i t y f o l l o w i n g b r i e f t r y p s i n treatment {19_ 28), although the trend was reversed over longer treatment perioïïs. The d i s t i n c t l y d i f f e r e n t behavior of the phenol-type h e r b i c i d e s f o l l o w i n g t r y p s i n treatment suggests that d i f f e r e n t d e t e r minants within the PS II protein complex e s t a b l i s h the "domains" that r e g u l a t e the binding p r o p e r t i e s of these i n h i b i t o r s . In s p i t e of the f a c t that phenol-type h e r b i c i d e s w i l l d i s p l a c e bound r a d i o l a b e l e d h e r b i c i d e s such as d i u r o n , these i n h i b i t o r s show noncompetitive i n h i b i t i o n (29, 30). At present, there are three l i n e s of evidence which favor Tfie involvement of two domains within the PS II complex that p a r t i c i p a t e in c r e a t i n g the binding s i t e s f o r these h e r b i c i d e s : (a) i s o l a t e d PS II p a r t i c l e s can be s e l e c t i v e l y depleted of a polypeptide with p a r a l l e l loss of a t r a zine s e n s i t i v i t y , but not dinoseb i n h i b i t i o n a c t i v i t y (33); (b) in r e s i s t a n t weed b i o t y p e s , c h i o r o p l a s t membranes that e x h i b i t extreme t r i a z i n e r e s i s t a n c e have increased s e n s i t i v i t y to the phenol-type h e r b i c i d e s (13); and (c) experiments with azido (photoa f f i n i t y ) d e r i v a t i v e s of phenol and t r i a z i n e h e r b i c i d e s r e s u l t in the covalent l a b e l i n g of d i f f e r e n t PS II polypeptides (20, 31). 9

I d e n t i f i c a t i o n of the T r i a z i n e Binding S i t e . An ultimate goal f o r the biochemical understanding of h e r b i c i d a l e f f e c t s on c h i o r o p l a s t membranes is to i d e n t i f y the s p e c i f i c membrane cons t i t u e n t that serves as h e r b i c i d e r e c e p t o r . For the s - t r i a z i n e s , i d e n t i f i c a t i o n of the s p e c i f i c receptor i s r e q u i r e d for understanding t r i a z i n e r e s i s t a n c e at the molecular l e v e l . Because t r y p s i n m o d i f i c a t i o n of membranes r e s u l t s in p r o t e i n - s p e c i f i c a l t e r a t i o n s of the membrane, the stepwise loss of h e r b i c i d e binding a f f i n i t y and binding s i t e s is a t t r i b u t e d to the stepwise a l t e r a t i o n of a protein that serves as the h e r b i c i d e r e c e p tor. One approach to the i d e n t i f i c a t i o n of h e r b i c i d e receptors has been the a n a l y s i s of peptide a l t e r a t i o n s f o l l o w i n g t r y p s i n treatment by using SDS-PAGE. A n a l y s i s of polypeptide a l t e r a t i o n in t h y l a k o i d membranes has been u n s a t i s f a c t o r y , however, because of the m u l t i p l i c i t y of p r o t e i n changes brought about by t r y p s i n (19). However, c h a r a c t e r i z a t i o n of membranes subfractionated f o l l o w i n g detergent treatment has been somewhat more i n f o r m a t i v e . When PS II enriched s u b f r a c t i o n s are i s o l a t e d from t r y p s i n treated membranes, the number of a l t e r e d polypeptides that could


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correspond to an a l t e r e d receptor p r o t e i n i s narrowed down to four candidates. Among these i s a polypeptide of 32 kDal (19). Highly p u r i f i e d PS II complexes i s o l a t e d by detergent f r a c t i o n a t i o n also contain a polypeptide of 32 kDal (32, 33). Trypsin, treatment of i s o l a t e d PS II complexes r e s u l t s in the degradation of the 32 kDal polypeptide with concomitant l o s s of h e r b i c i d e a c t i v i t y (32, 33). F i n a l l y , s e l e c t i v e removal of the 32 kDal polypeptide from PS II p a r t i c l e s by a d d i t i o n a l detergent t r e a t ments r e s u l t s in loss of both diuron and a t r a z i n e binding (33). These l i n e s of evidence a l l i m p l i c a t e a 32 kDal polypeptide as the receptor f o r t r i a z i n e and urea c l a s s e s of h e r b i c i d e s . The a s s o c i a t i o n of diuron and a t r a z i n e with c h i o r o p l a s t membranes i s v i a a h i g h - a f f i n i t y , but noncovalent b i n d i n g . Attempts at p h y s i c a l i s o l a t i o n of proteins labeled by r a d i o a c t i v e i n h i b i t o r s have f a i l e d because techniques such as detergent f r a c t i o n a t i o n or e l e c t r o p h o r e t i c separation r a p i d l y lead to a new e q u i l i b r i u m and the d i s s o c i a t i o n of the noncovalent r e c e p t o r - i n h i b i t o r complex. One approach that overcomes t h i s d i f f i c u l t y in i d e n t i f y i n g a h e r b i c i d e receptor i s to attach a r a d i o labeled p h o t o a f f i n i t y azido d e r i v a t i v e of the h e r b i c i d e to i t s h i g h - a f f i n i t y receptor p r o t e i n . A c t i v a t i o n of the azido f u n c t i o n of p h o t o a f f i n i t y compounds by UV i r r a d i a t i o n produces a n i t r e n e that i s h i g h l y r e a c t i v e (34). If the compound remains l o c a l i z e d at i t s h i g h - a f f i n i t y s i t e throughout the l i f e t i m e of the d e s t a b i l ized n i t r e n e group, covalent binding w i l l occur at the binding site. A z i d o a t r a z i n e has been shown to i n h i b i t photosynthetic e l e c t r o n t r a n s p o r t at a s i t e i d e n t i c a l to that of a t r a z i n e (20). We have used a z i d o a t r a z i n e as a p h o t o a f f i n i t y probe to ident~T7y the h e r b i c i d e - r e c e p t o r p r o t e i n in c h l o r o p l a s t s of _A. hybridus. In order to demonstrate the s p e c i f i c i t y of binding to a h i g h a f f i n i t y s i t e , membranes from both s u s c e p t i b l e and r e s i s t a n t biotypes were u t i l i z e d . A n a l y s i s of membrane polypeptides from s u s c e p t i b l e and r e s i s t a n t membranes by SDS-PAGE i s shown in Figure 3 (Coomassie blue stained polypeptides are in lanes A ) . No major d i f f e r e n c e s in polypeptide composition or s t a i n i n g i n t e n s i t y between the two samples are apparent. For both c a s e s , membranes were UV i r r a d i a t e d in the presence of a z i d o - ^ C a t r a z i n e p r i o r to e l e c t r o p h o r e s i s . A n a l y s i s of the gel by a f l u o r o g r a p h i c technique showed no detectable bound r a d i o l a b e l a s s o c i a t e d with the membrane sample from r e s i s t a n t c h l o r o p l a s t s . In the s u s c e p t i b l e membranes, however, the r a d i o l a b e l extended from a region corresponding to 34 kDal to that of a stained polypeptide at 32 kDal. We s h a l l present evidence in the f o l l o w i n g d i s c u s s i o n that t h i s pattern of l a b e l i n g i s due to covalent h e r b i c i d e a s s o c i a t i o n to a s i n g l e p r o t e i n which e x i s t s in e i t h e r of two molecular weight s p e c i e s : a developmental precursor form of 34 kDal which is s i z e - p r o c e s s e d to a 32 kDal form. For the remaining d i s c u s s i o n we s h a l l designate t h i s t r i a z i n e h e r b i c i d e receptor as the 32-34 kDal polypeptide.


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Figure 3. Polyacrylamide slab gel elec trophoresis of thylakoid membrane poly peptides from susceptible and resistant biotypes of A. hybridus, stained for pro tein (lanes A) and by fluorography (lanes B). Susceptible and resistant membranes were incubated with 0.5 μΜ azido- Catrazine under UV light for 10 min prior to SDS solubilization. The predominant location of the radiolabel, as shown by fluorography, is over the 34- to 32-kDal size class. 14


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One a d d i t i o n a l l i n e of evidence that independently i m p l i c a t e s a 32 kDal polypeptide in t r i a z i n e binding comes from studies on c h l o r o p l a s t s i s o l a t e d from a maize mutant that s p e c i f i c a l l y lacks the s t a i n a b l e 32 kDal polypeptide and the r a p i d l y labeled 34 kDal polypeptide. C h l o r o p l a s t s of t h i s PS I I - l e s s l e t h a l mutant lack binding s i t e s f o r r a d i o a c t i v e a t r a z i n e (35). Step-wise M o d i f i c a t i o n of the Herbicide Binding Polypeptide In V i t r o . Evidence from a t r a z i n e binding studies described in a previous s e c t i o n suggested that trypsin-mediated a l t e r a t i o n s of surface exposed membrane polypeptides r e s u l t e d i n a sequential a l t e r a t i o n of a t r a z i n e binding s i t e s . F i r s t , a rapid alteration of h e r b i c i d e a f f i n i t y was detected followed by a more gradual loss of d e t e c t a b l e binding s i t e s . This stepwise a l t e r a t i o n was f u r t h e r i n v e s t i g a t e d at the l e v e l of polypeptide s t r u c t u r e by using the s e l e c t i v e membrane m o d i f i e r , t r y p s i n , against membranes that had been c o v a l e n t l y tagged with the r a d i o l a b e l e d photoa f f i n i t y t r i a z i n e probe. As shown in Figure 4A, the use of low c o n c e n t r a t i o n s of a z i d o - ^ C - a t r a z i n e r e s u l t e d in l a b e l i n g of polypeptide species at 34 kDal. We i n t e r p r e t these data as i n d i c a t i n g that the 34 kDal form of the 32-34 kDal polypeptide creates the h i g h - a f f i n i t y h e r b i c i d e binding s i t e . In membrane samples treated with 2 yg trypsin/ml f o r 15 min (see legend of Figure 4 f o r d e t a i l s ) , a t r y p s i n concentration shown p r e v i o u s l y to p r i n c i p a l l y bring about changes in a t r a z i n e a f f i n i t y (Figure 2B), the 34 kDal polypeptide l a b e l e d with a z i d o a t r a z i n e was a l t e r ed to a species that comigrated with a stained 32 kDal polypept i d e (Figure 4B). At higher t r y p s i n concentrations (Figures 4C and 4D; 10 and 40 ug t r y p s i n / m l , r e s p e c t i v e l y ) where loss of both e l e c t r o n t r a n s p o r t f u n c t i o n and i n h i b i t o r binding s i t e s were observed p r e v i o u s l y , the polypeptide tagged by a z i d o a t r a z i n e was f u r t h e r degraded to species at 18 and then 16 kDal in a s e q u e n t i a l , stepwise manner. [The experiment of Figure 4 u t i l i z e d membranes which were f i r s t l a b e l e d with a z i d o a t r a z i n e and then subjected to t r y p s i n treatment.] We conclude that the major covalent attachment s i t e f o r a z i d o a t r a z i n e is in a hydrophobic region of the membrane which is i n a c c e s s i b l e to t r y p s i n , thereby leaving an i n t r i n s i c 16 kDal fragment of the t r i a z i n e binding p r o t e i n associated with the membrane f o l l o w i n g t r y p s i n treatment. Further degradation of the 16 kDal polypeptide is not observed a f t e r prolonged t r y p s i n treatment. The data of Figure 4 i n d i c a t e d that a 34 kDal form of the 32-34 kDal polypeptide i s the h i g h - a f f i n i t y binding s i t e f o r triazines. To t e s t whether the t r y p s i n - d e r i v e d , membrane-bound fragments of t h i s p r o t e i n s t i l l bound h e r b i c i d e , a z i d o a t r a z i n e was used against t r y p s i n - t r e a t e d membranes. Figure 5 shows the fluorogram of e l e c t r o p h o r e t i c a l l y separated pea c h i o r o p l a s t polypeptides from c o n t r o l (A and B) and t r y p s i n - t r e a t e d (C and D) membranes that were tagged with a z i d o a t r a z i n e a f t e r the protease m o d i f i c a t i o n . When a z i d o a t r a z i n e was applied against


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Figure 4. Fluorogram of a polyacrylamide gel that shows trypsin sensitivity of the 34-kDal polypeptide of pea chioro plast membranes following covalent radiolabeling with azido- C-atrazine. Key: A, control; B, 2 μg trypsin/mL; C, 10 μ-g trypsin/mL; D, 40 pg trypsin/mL for 15 min at room temperature. Proteolysis was stopped by the addition of a 20-fold excess of trypsin inhibitor. Membranes were washed twice in PSNM buffer prior to electrophoresis. 14

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Figure 5. Fluorogram of electrophoretically separated pea chioroplast poly peptides from control and trypsin-treated membranes that were tagged with radio labeled azidoatrazine. Key: A, control membranes (50 fig Chl/mL) UV-irradi ated 10 min in the presence of 2.5 μΜ azido- C-atrazine; B, as in A, but with 25 μΜ azidoatrazine; C, treatment of chloroplasts with 2 μg trypsin/mL for 15 min prior to tagging with 25 μΜ azidoatrazine; D, as in C, but 40 μg tryp sin/mL for 15 min prior to tagging with azidoatrazine. 14


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c o n t r o l membranes at a concentration of 2.5 uM (equivalent to an a t r a z i n e concentration r e q u i r e d to i n h i b i t e l e c t r o n t r a n s p o r t by 90%) o n l y the 34 kDal polypeptide species was labeled (Figure 5A). When a 1 0 - f o l d higher concentration is u t i l i z e d (25 y M ) , equivalent to an a t r a z i n e concentration r e q u i r e d to i n h i b i t e l e c t r o n t r a n s p o r t by 90% in t r y p s i n - t r e a t e d membranes) both the 34 and 32 kDal forms of the 32-34 kDal polypeptide are labeled (Figure 5B). Mild t r y p s i n - t r e a t m e n t (2 yg t r y p s i n / m l , 15 min) followed by a z i d o a t r a z i n e tagging using 25 yM a z i d o a t r a z i n e (Figure 5C) r e s u l t e d in tagging of the 32 kDal species ( i n c o n t r a s t to l a b e l ing both the 34 and 32 kDal forms of the polypeptide in c o n t r o l membranes). This supports the hypothesis that the presence of the t r y p s i n - d e r i v e d 32 kDal polypeptide provides both an a f f i n i t y s i t e and a binding s i t e f o r t r i a z i n e s . When thylakoids are tagged f o l l o w i n g high t r y p s i n treatment (40 yg t r y p s i n / m l , 15 min) no label is observed at 32 kDal, nor at the molecular weights of the expected breakdown products of 18 and 16 kDal. These data f u r t h e r suggest that a f f i n i t y and/or binding to the i n t r i n s i c fragments alone can not occur. It should be noted that in a l l cases where a high concentration of a z i d o a t r a z i n e i s u t i l i z e d (25 yM, Figures 5B, C, and D) a d e t e c t a b l e l e v e l of n o n s p e c i f i c binding of the compound to a number of t h y l a k o i d polypeptides i s observed. The most i n t e n s e l y l a b e l e d was a p o l y peptide species of 25 kDal before t r y p s i n treatment or 23 kDal a f t e r protease d i g e s t i o n . This polypeptide was p r e v i o u s l y demon s t r a t e d to be the apoprotein of the l i g h t - h a r v e s t i n g c h l o r o p h y l l a/b pigment p r o t e i n which has a surface-exposed segment (23). The T r i a z i n e Receptor P r o t e i n i s a Product of C h l o r o p l a s t Directed P r o t e i η Synthesfs"] T r i a z i n e r e s i s t a n c e has been demon s t r a t e d from r e c i p r o c a l c r o s s i n g experiments to be i n h e r i t e d u n i p a r e n t a l l y through the female parent i n J3. campestri s (16). As the r e s i s t a n c e mechanism has been shown to r e s i d e at the l e v e l of the c h i o r o p l a s t membrane, a r o l e f o r the c h i o r o p l a s t genome i n c o n f e r r i n g r e s i s t a n c e is strongly implied (17). It was of i n t e r e s t to determine i f the c h i o r o p l a s t membrane p r o t e i n of 32-34 kDal that binds the p h o t o a f f i n i t y t r i a z i n e , and which appears to be required f o r t r i a z i n e binding in i s o l a t e d PS II p a r t i c l e s , i s a c h i o r o p l a s t gene product. In developing c h l o r o p l a s t s , in p a r a l l e l to the appearance of f u n c t i o n a l a c t i v i t i e s , there i s r a p i d synthesis and accumulation of a major t h y l a koid p r o t e i n of 34 kDal (36). This r a p i d l y synthesized c h i o r o p l a s t p r o t e i n has been shown to be encoded by the c h i o r o p l a s t genome i n 1. mays (37). This s e c t i o n o u t l i n e s experiments that were c a r r i e d out to determine i f the c h i o r o p l a s t polypeptide, which serves as the t r i a z i n e binding s i t e , i s i d e n t i c a l to the chloroplast-encoded p r o t e i n of the same molecular weight. As was shown i n Figure 4, the 34 kDal polypeptide l a b e l e d by a z i d o a t r a z i n e shows a s p e c i f i c stepwise a l t e r a t i o n mediated



50

by the p r o t e o l y t i c enzyme t r y p s i n . We i n t e r p r e t that the s p e c i f i c stepwise a l t e r a t i o n o f t h i s polypeptide is regulated by (a) the presence of l y s i n e or a r g i n i n e residues in the primary sequence of t h i s peptide that are s t e r i c a l l y a c c e s s i b l e to the p r o t e o l y t i c probe and (b) the increased a c c e s s i b i l i t y of s p e c i f i c cleavage s i t e s caused by trypsin-mediated a l t e r a t i o n s of other surface-exposed peptides that share a common microenvironment at the membrane surface with the 32-34 kDal polypeptide. The s t e p wise t r y p s i n degradation of the 34 kDal form of the polypeptide to 32, 18,and 16 kDal d e f i n e s a "map" of i t s s t r u c t u r a l i n t e g r a t i o n within the membrane and can be used to i d e n t i f y the same p r o t e i n "tagged" by an independent method. The c h l o r o p l a s t - s y n t h e s i z e d p r o t e i n of the same molecular weight as the t r i a z i n e - b i n d i n g protein i s the most r a p i d l y synt h e s i z e d p r o t e i n in vivo of pea t h y l a k o i d membranes. Thus, i t s t r y p s i n s e n s i t i v i t y can be r e a d i l y evaluated by autoradiographic techniques. Pea seedlings were allowed to take up and i n c o r p o r ate 3 5 s _ e t h i o n i n e f r 4 h; t h i s r e s u l t e d in r a d i o l a b e l i n g of r a p i d l y synthesized p o l y p e p t i d e s . Control and t r y p s i n - t r e a t e d membrane samples were subjected to SDS-PAGE. Following s t a i n i n g for p r o t e i n , polypeptides were analyzed for r a d i o l a b e l i n c o r p o r a t i o n by X-ray fluorography. As shown in Figure 6A, i n c o r p o r a t i o n of 3 5 $ - r a d i o l a b e l was observed f o r two major molecular weight species — the apop r o t e i n of the l i g h t harvesting complex (LHC) at 25 kDal and a polypeptide of 34 kDal. Following t r y p s i n treatment (Figure 6B), the r a d i o l a b e l associated with the LHC polypeptides was a l t e r e d in e l e c t r o p h o r e t i c m o b i l i t y by 2 kDal f o l l o w i n g the expected a l t e r a t i o n f o r the Coomassie-stained peptide species ( 2 3 ) . The r a p i d l y synthesized 34 kDal polypeptide was also s u s c e p t i b l e to trypsin. Corresponding to i t s loss with t r y p s i n treatment, new r a d i o l a b e l e d bands appeared at 32 (Figure 6B), then 18, and 16 kDal (Figures 6C,D) i n an i d e n t i c a l , t r y p s i n - c o n c e n t r a t i o n dependent fashion to that of the p h o t o a f f i n i t y tagged 34 kDal p o l y peptide (see Figure 4 ) . From the i d e n t i c a l t r y p s i n s e n s i t i v i t y of the p h o t o a f f i n i t y tagged polypeptide and the r a p i d l y synthes i z e d c h i o r o p l a s t p r o t e i n of the same molecular weight, we conclude that the two polypeptides are one and the same. m

0

Evidence that the T r i a z i n e Binding Protein i s Present in r i a z i n e Resistant Weed B i o t y p e s . The u t i l i z a t i o n of a photoa f f i n i t y labeled t r i a z i n e h e r b i c i d e has been i n v a l u a b l e in the d e f i n i t i v e i d e n t i f i c a t i o n of one s p e c i f i c polypeptide of the PS II complex as the t r i a z i n e binding s i t e . The absence of covalent l a b e l i n g in r e s i s t a n t membranes suggests that e i t h e r the polypeptide i s missing from the membrane or that i t i s present, but g e n e t i c a l l y a l t e r e d , r e s u l t i n g in an a l t e r a t i o n in i t s primary s t r u c t u r e , and p o s s i b l y changing i t s conformation in the membranes. In s u s c e p t i b l e membranes, t r i a z i n e and urea h e r b i c i d e s com-


3. STEINBACK ET AL.


51

Figure 6. Fluorogram of polyacrylamide gel showing trypsin sensitivity of the 34-kDal polypeptide of pea chioro plast membranes following in vivo incor poration of S-methionine in whole leaves. Key: A, control; B, 2 ^g trypsin/ mL; C, 10 ng trypsin/mL; D, 40 μg trypsin/mL. Treatments were as described in Figure 4. 35


52


pete f o r the same binding region as determined by d i r e c t compet i t i o n s t u d i e s (9). In r e s i s t a n t membranes, whereas t r i a z i n e s e n s i t i v i t y i s e x t e n s i v e l y d i m i n i s h e d , the a b i l i t y of diuron to i n h i b i t e l e c t r o n t r a n s p o r t i s not a l t e r e d s i g n i f i c a n t l y (3., 9., 13). This i n d i c a t e s an a l t e r a t i o n in the t r i a z i n e , but not the urea a f f i n i t y s i t e and f u r t h e r suggests that a common binding s i t e , i . e . , p r o t e i n , i s present in r e s i s t a n t membranes, but possesses an a l t e r e d a f f i n i t y f o r the t r i a z i n e h e r b i c i d e s . It was of i n t e r e s t to determine i f the polypeptide r e s p o n s i b l e for h e r b i c i d e s e n s i t i v i t y i n c h l o r o p l a s t s of normal, t r i a z i n e - s u s c e p t i b l e plants was also present, i . e . , s y n t h e s i z e d , in the r e s i s t a n t biotypes. Using the techniques f o r 3 5 $ - e t h i o n i n e i n c o r p o r a t i o n j j i v j v o described f o r peas, i n c o r p o r a t i o n of ^ S - r a d i o label i n t o t h y l a k o i d polypeptides of s u s c e p t i b l e and r e s i s t a n t biotypes of A. hybridus was i n v e s t i g a t e d . The data shown in Figure 7 demonstrate that the 34 kDal polypeptide i s synthesized in both h e r b i c i d e - s u s c e p t i b l e and r e s i s t a n t p l a n t s . Furthermore, the polypeptide in r e s i s t a n t membranes shows an i d e n t i c a l s e n s i t i v i t y to t r y p s i n - t r e a t m e n t as the 34 kDal polypeptide of the s u s c e p t i b l e membranes. Whereas i t is evident that the polypept i d e i s synthesized and present in both b i o t y p e s , there i s no apparent s i z e d i f f e r e n c e or change in membrane o r i e n t a t i o n , as r e f l e c t e d by t r y p s i n s e n s i t i v i t y , that can account for the extreme d i f f e r e n c e s in t r i a z i n e a f f i n i t i e s . The t r i a z i n e - r e s i s t a n c e mechanism, t h e r e f o r e , probably r e s i d e s in an a l t e r e d primary s t r u c t u r e of t h i s p r o t e i n . m

5

Acknowledgements: This research was supported, in p a r t , by a Grant from the United States - I s r a e l B i n a t i o n a l A g r i c u l t u r a l Research and Development Fund (BARD), DOE Contract DE-AC02-7ER01338 to Michigan State U n i v e r s i t y , and a Wellesley College F a c u l t y Aid Grant to K. Steinback. We also thank Dr. Gary Gardner of S h e l l A g r i c u l t u r a l Chemicals f o r his c o l l a b o r a t i o n and c o n s u l t a t i o n s in experiments using a z i d o - a t r a z i n e .


STEINBACK ET AL.


Figure 7. Fluorogram of a polyacrylamide gel showing trypsin sensitivity of the 34-kDal polypeptide of chioroplast thylakoid membranes isolated from susceptible (S) and resistant (R) biotypes of A. hybridus following in vivo incorporation of S-methionine in whole leaves. Isolated thylakoid membranes were treated with A, no trypsin; B, 2 fig trypsin/mL; C, 20 μg trypsin/mL for 15 min as described for Figure 4. 35


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Literature Cited 1. Ashton, F. M.; Crafts, A. S. "Mode of Action of Herbicides"; Wiley: New York, 1973; pp. 69-99. 2. Wright, K.; Corbett, J. Κ. Z. Naturforsch. 1979, 34c, 966-72. 3. Pfister, K.; Arntzen, C. J. Z. Naturforsch. 1979, 34c, 996-1009. 4. Bouges-Bocquet, B. Biochim. Biophys. Acta 1973, 314, 250-6. 5. Velthuys, B. R.; Amesz, J. Biochim. Biophys. Acta 1974, 333, 85-94. 6. Tischer, W.; Strotmann, H. Biochim. Biophys. Acta 1977, 460, 113-25. 7. Trebst, Α.; Draber, W. "Advances in Pesticide Research"; Geissbuhler, H., Ed.; Pergamon Press: Oxford, 1979; pp. 223-34. 8. Strotmann, H.; Tischer, W.; Edelman, K. Ber. Deutsch Bot. Ges. 1974, 87, 457-63. 9. Pfister, K.; Radosevich, S. R.; Arntzen, C. J. Plant Physiol. 1979, 64, 995-9. 10. Radosevich, S. R.; DeVilliers, O. T. Weed Sci., 1976, 24, 229-32. 11. Souza-Machado, V.; Arntzen, C. J.; Bandeen, J. D.; Stephen son, G. R. Weed Sci., 1978; 26, 318-22. 12. Arntzen, C. J.; Ditto, C. L.; Brewer, P. Proc. Natl. Acad. Sci., U.S.A., 1979, 76, 278-82. 13. Pfister, K.; Watson, J.; Arntzen, C. J. Weed Sci. 1981 (in press). 14. Bowes, J.; Crofts, A. R.; Arntzen, C. J. Arch. Biochem. Biophys. 1980, 200, 303-8. 15. Arntzen, C. J.; Pfister, K.; Steinback, Κ. E. "Studies of Herbicide Resistance", LeBaron, H.; Gressel, J . , Eds.; CRC Press (in press). 16. Souza-Machado, V.; Bandeen, J. D.; Stephenson, G. R.; Lavigne, P. Can J. Plant Sci. 1978, 58, 977-81. 17. Darr, S.; Souza Machado, V.; Arntzen, C. J. Biochim. Biophys. Acta 1981, 634, 219-28. 18. "Blair, G. E.; Ellis, R. J. Biochim. Biophys. Acta 1973, 319, 223-34. 19. Steinback, K.E.; Pfister, K.; Arntzen, C. J. Z. Naturforsch. 1981, 36c, 98-108. 20. Pfister, K.; Steinback, Κ. E.; Gardner, G.; Arntzen, C. J. Proc. Natl. Acad. Sci., U.S.A. 1981, 78, 981-5. 21. Gardner, G. Science 1981, 211, 937-40. 22. Laemmli, U. K. Nature (London) 1970, 227, 680-5. 23. Steinback, Κ. E.; Burke, J. J.; Arntzen, C. J. Arch. Biochem. Biophys. 1979, 195, 546-57. 24. Lasky, R. Α.; Mills, A. D. Eur. J. Biochem. 1975, 56, 335-41. 25. Regitz, G.; Ohad, I. "Proc. 3rd. Int. Congr. on Photosynthe sis", Vol. III; Avron, M., Ed.; Elsevier: Amsterdam, 1975; p. 1615.


3.

STEINBACK ET AL.

26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.


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Renger, G. Z. Naturforsch. 1979, 34c, 1010-14. Trebst, A. Z. Naturforsch. 1979, 34c, 986-91. Böger, P.; Kunert, K. J. Z. Naturforsch. 1979, 34c, 1015-25. Reimer, S.; Link, K.; Trebst, A. Z. Naturforsch. 1979, 34c, 419-26. Oettmeier, W.; Masson, K. Pestic. Biochem. Physiol. 1980, 14, 86-97. Oettmeier, W.; Masson, K.; Johanningmeier, U. FEBS Lett. 1980, 118, 267-70. Croze, E.; Kelly, M.; Horton, P. FEBS Lett. 1979, 103, 22-6. Mullet, J. E.; Arntzen, C. J. Biochim. Biophys. Acta 1981, 635, 236-48. Bayley, H.; Knowles, J. R. "Methods in Enzymology", Vol. XLVI; Jackoby, W. B.; Wilchek, M., Eds.; Academic Press: New York, 1977; 69-114. Leto, K.; Keresztes, Α.; Arntzen, C. J. Plant Physiol. 1981, (in press). Grebanier, A. E.; Steinback, Κ. E.; Bogorad, L. Plant Physiol. 1979, 63, 436-9. Bedbrook, J. R.; Link, G.; Coen, D. M.; Bogorad, L.; Rich, A. Proc. Natl. Acad. Sci., U.S.A. 1978, 75, 3060-4.

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Biochemical Responses Induced by Herbicides - American Chemical

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